NTBC is a drug marketed by Swedish Orphan Biovitrum International AB under the brand name Orfadin® and it is used to slow the effects of hereditary tyrosinemia type 1 (HT-1) in adult and pediatric patients. It has been approved by FDA and EMA in January 2002 and February 2005 respectively.
HT-1 disease is due to a deficiency of the final enzyme of the tyrosine catabolic pathway fumarylacetoacetate hydrolase. NTBC is a competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme which precedes film arylacetoacetate hydrolase. By inhibiting the normal catabolism of tyrosine in patients with HT-1, NTBC prevents the accumulation of the toxic intermediates maleylacetoacetate and fumarylacetoacetate, that in patients with HT-1 are converted to the toxic as succinylacetone and succinylacetoacetate, the former inhibiting the porphyrin synthesis pathway leading to the accumulation of 5-aminolevulinate.
Usefulness of NTBC in the treatment of further diseases has also been documented. A non-comprehensive list is reported hereinafter.
Effectiveness of Orfadin® in the treatment of diseases where the products of the action of HPPD are involved (e.g., HT-1) has been described notably in EP0591275131 corresponding to U.S. Pat. No. 5,550,165B1. Synthesis of NTBC is also described in this patent.
WO2011106655 reports a method for increasing tyrosine plasma concentrations in a subject suffering from oculocutaneous/ocular albinism, the method comprising administering to the subject a pharmaceutically acceptable composition comprising NTBC in the range of between about 0.1 mg/kg/day to about 10 mg/kg/day.
U.S. Pat. No. 8,354,451B2 reports new methods of combating microbial infections due to fungi or bacteria by means of administration to a subject of a therapeutically active amount of NTBC.
WO2010054273 discloses NTBC-containing compositions and methods for the treatment and/or prevention of restless leg syndrome (RLS).
EP1853241B1 claims the use of NTBC in the treatment of a neurodegenerative disease, notably Parkinson disease.
Introne W. J., et al., disclosed usefulness of nitisinone in the treatment of alkaptonuria (Introne W. J., et al., Molec. Genet. Metab., 2011, 103, 4, 307). The key step of the synthesis reported in EP0591275B1 (now propriety of Swedish Orphan Biovitrum International AB, SE), involves the reaction of 2-nitro-4-trifluoromethylbenzoyl chloride and cyclohexane-1,3-dione in the presence of triethylamine and then use of acetone cyanohydrin in order to promote the rearrangement of the key intermediate enol ester. After washing and extraction from CH2Cl2, the crude product is recrystallized from ethyl acetate to get the desired 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione as a solid having a melting point of 88-94° C.
Another patent (U.S. Pat. No. 4,695,673) filed in name of Stauffer Chemical Company disclosed a process of synthesis of acylated 1,3-dicarbonyl compounds in which the intermediate enol ester is isolated prior to its rearrangement into the final product, said rearrangement making use of a cyanohydrin compound derived from alkali metal, methyl alkyl ketone, benzaldehyde, cyclohexanone, C2-C5 aliphatic aldehyde, lower alkyl 341 or directly by using hydrogen cyanide.
Yet another patent (U.S. Pat. No. 5,006,158) filed in name of ICI Americas Inc. disclosed a process similar to the one disclosed in U.S. Pat. No. 4,695,673 wherein the intermediate enol ester was isolated prior to its rearrangement into the final product by use of potassium cyanide. Said reaction can optionally be done by concomitant use of a phase transfer catalyst such as Crown ethers. The preferred solvent for conducting such a reaction is 1,2-dichloroethane.
Still a further patent (EP0803791) filed in name of Zeneca Ltd disclosed an alternative synthesis of nitisinone involving the reaction of 1,3-cyclohexanedione and variously substituted benzoyl chloride in the presence of sodium or potassium carbonate in CH3CN or DMF. Best yields were obtained using CH3CN as solvent and sodium carbonate as the base. Reaction was performed at 55-57° C. in 17 hours.
It is well known that one of the problems of the actual drug formulation (i.e., Orfadin® capsules) is its chemical instability. Indeed, even if Orfadin® has to be stored in a refrigerator at a temperature ranging from 2° C. to 8° C., its shelf life is of only 18 months. After first opening, the in-use stability is a single period of 2 months at a temperature not above 25° C., after which it must be discarded. It will be evident that such storage conditions have an impact in the distribution chain of the medicine, in terms of costs and also in terms of logistics for the patient. Therefore, there is an urgent need of more stable formulations, both from a logistic supply chain point of view, and from the patient compliance point of view. Since the formulation of Orfadin® contains only the active ingredient and starch as excipient, relative instability may be attributed to the active pharmaceutical ingredient itself; in other words it can derive from the way it is synthesized and/or the way it is extracted from the reaction mixture, and/or the way it is finally crystallized. Furthermore, some impurities may contribute to render the final product less stable overtime. Consequently, it is of major importance to identify a process of synthesis and/or a crystallization method that enable the reliable production of a highly pure and stable product.
Impurities as herein-above mentioned can derive either from the final product itself (through chemical degradation) or directly from the starting materials/solvents used in the process of synthesis. Regarding the latter option, it is therefore primordial to ascertain that at each step, impurities are completely removed in order not to get them at the final stage, also considering that some of them could potentially be cyto/genotoxic.
The impurities correlated to nitisinone can be either derived from the starting materials themselves (i.e., impurities 1 and 2) or obtained as side products during the process of synthesis major under storage conditions (i.e., impurities 3 to 5) and are the following:    2-nitro-4-(trifluoromethyl) benzoic acid (Impurity n° 1),    1,3-cyclohexanedione (CHD) (Impurity n° 2),    4-(trifluoromethyl)salicylic acid (Impurity n° 3),    2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (Impurity n° 4), and    6-trifluoromethyl-3,4-dihydro-2H-xanthene-1,9-dione (Impurity n° 5).

Impurity-2, impurity-3, and impurity-5 have been previously reported in WO2015101794. Strangely, impurity-4 has never been reported, even if it is an obvious side-product which can easily be formed during the coupling reaction between 1,3-cyclohexanedione and 2-nitro-4-(trifluoromethyl) benzoic acid, the latter being not 100% pure but Wining some amount of regioisomer 3-nitro-4-(trifluoromethyl) benzoic acid.
Potential genotoxicity of impurity n° 4 which possesses an aromatic nitro moiety was assessed using in-silica techniques and resulted to be a potential genotoxic impurity. According to the FDA ICH M7 guidelines, daily intake of a mutagenic impurity (Threshold of Toxicological Concern, TTC) in an amount not greater than 1.5 μg per person is considered to be associated with a negligible risk to develop cancer over a lifetime of exposure. Consequently, assuming a daily dose of 2 mg/kg, for a person weighing 70 kg, the maximum tolerated impurity content of such a compound would be of about 11 ppm, as calculated according to the equation underneath.
      concentration    ⁢                  ⁢    limit    ⁢                  ⁢          (      ppm      )        =            TTC      ⁢                          ⁢              (                  µ          ⁢                                          ⁢          g          ⁢                      /                    ⁢          day                )                    Dose      ⁢                          ⁢              (                  g          ⁢                      /                    ⁢          day                )            
It is therefore of paramount importance to ensure that the process of synthesis of nitisinone and the purification steps of the same give rise to an API devoid of such impurity n° 4, or at least far below the threshold of 11 ppm as indicated above. The skilled person will understand that total absence of said impurity is highly desirable.
It is well known in the pharmaceutical field that investigation of potential polymorphism of a solid API is of crucial importance and is also recommended by major regulatory authorities such as FDA.
Notwithstanding the fact that nitisinone has been used for years to treat HT-1 patients, it appears that no NTBC formulation fully satisfies the requisites of stability and/or compliance standard for the patients. Therefore, there is an unmet medical need of long-term pure and stable formulations.